<em>In Situ</em> Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

School of Dentistry and Dental Research Institute, Seoul National University

Periodontitis is a polymicrobial disease that arises from the dysbiosis of the plaque biofilm. To study polymicrobial interactions with gingival epithelial cells, the oral commensal Fusobacterium nucleatum and the periodontal pathogen Treponema denticola were chosen due to their opposing effects on the expression of human beta-defensins (HBDs) and interleukin (IL)-8 in gingival epithelial cells. Immortalized gingival epithelial HOK-16B cells were infected with either F. nucleatum or T. denticola alone or together, and the expression of HBDs and IL-8 was investigated. Coinfection with F. nucleatum and T. denticola neutralized the stimulatory and suppressive effects on the expression of HBD-2 and -3, but the suppressive effect of T. denticola on IL-8 expression remained. In CHO/CD14/TLR2 reporter cells, T. denticola attenuated F. nucleatum-induced activation of TLR2, a receptor that mediates HBD induction. Although F. nucleatum facilitated the invasion of T. denticola into host cells, T. denticola interfered with the fusion of internalized F. nucleatum with lysosomes, which may avert TLR9-dependent IL-8 induction. Furthermore, T. denticola suppressed the F. nucleatum-stimulated accumulation of intracellular reactive oxygen species (ROS), a group of essential signaling molecules for the TLR2 and TLR9 pathways. The elimination of ROS using N-acetyl cysteine completely blocked the inductions of HBD-3 and IL-8 and significantly reduced HBD-2 induction by F. nucleatum, confirming the importance of ROS in the host response. In sum, T. denticola incapacitates the F. nucleatum-induced expression of HBDs and IL-8 in gingival epithelial cells by interrupting endo-lysosomal maturation and ROS-dependent TLR activation. These results may provide new insights into polymicrobial interactions in the gingival sulcus.

A periodontal pathogen Treponema denticola hijacks the Fusobacterium nucleatum-driven host response.

Gingival fibroblasts (GFs) are an important regulatory cell type in the progression of periodontitis. This study aimed to compare the expression levels of genes associated with inflammation, extracellular matrix degradation and bone destruction in GFs isolated from healthy and periodontitis subjects in the absence and presence of Porphyromonas gingivalis.

Gingival fibroblasts from periodontitis patients exhibit inflammatory characteristics in vitro.

The purpose of this study was to investigate the mechanism(s) of interleukin (IL)-8 suppression by Treponema denticola, one of the major periodontal pathogens, in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with wild-type (WT), dentilisin-deficient (K1) or flagellin-deficient (flgE) T. denticola in the presence or absence of 2% human serum for 24 h. The levels of IL-8 expression were measured with real-time reverse transcription PCR and ELISA. In the absence of human serum, the WT and flgE, but not K1, substantially reduced not only the levels of IL-8 protein but also of IL-8 mRNA. Such downregulation of IL-8 mRNA was independent of bacterial invasion. Degradation of cytokine mixture by the WT, K1 and flgE revealed dentilisin-dependent preferential degradation of tumor necrosis factor (TNF)-α, an IL-8-inducing cytokine. WT and flgE significantly decreased the levels of TNFα secreted by HOK-16B cells, suggesting modulation of IL-8 through dentilisin-mediated degradation of TNFα. The addition of human serum to the culture potentiated the suppressive effect of T. denticola, resulting in substantial reductions of IL-8 and TNFα levels, even by K1. The serum-dependent effects of T. denticola were attributed to its ability to suppress the accumulation of intracellular reactive-oxygen species (ROS), a group of ubiquitous signaling molecules. Pretreatment with an antioxidant suppressed TNFα-induced IL-8 expression, confirming the role of ROS in TNFα signaling. Collectively, T. denticola targeted a key inflammatory cytokine and its signaling molecule to modulate the host innate immune response, which provides a new insight into modulation of host immunity by a periodontal pathogen.

Mechanisms of IL-8 suppression by Treponema denticola in gingival epithelial cells.

We previously reported that human serum significantly reduces the invasion of various oral bacterial species into gingival epithelial cells in vitro. The aims of the present study were to characterize the serum component(s) responsible for the inhibition of bacterial invasion of epithelial cells and to examine their effect on periodontitis induced in mice.

N-acetylcysteine and the human serum components that inhibit bacterial invasion of gingival epithelial cells prevent experimental periodontitis in mice.

Porphyromonas gingivalis is one of the well-characterized periodontal pathogens involved in periodontitis. The invasive and proteolytic activities of P. gingivalis clinical isolates have been shown to be associated with heterogenic virulence, as determined in a mouse abscess model. The aims of the present study were to identify a P. gingivalis strain with a low virulence among clinical isolates, based on its invasive ability and cytokine proteolytic activities, and to explore the preferential degradation of a certain cytokine by P. gingivalis. P. gingivalis ATCC 33277, W50, and 10 clinical isolates were used. After incubating bacteria with IL-4, IL-6, IL-10, IL-17A, TNFα, IFNγ, and IL-1α, the amounts of remaining cytokines were determined by ELISA. Invasion ability was measured by a flow cytometric invasion assay. There was inter-strain variability both in the cytokine proteolytic activities and invasion ability. In addition, differential degradation of cytokines by P. gingivalis was observed: while IFNγ and IL-17A were almost completely degraded, inflammatory cytokines TNFα and IL-1α were less susceptible to degradation. Interestingly, the invasion index, but not cytokine proteolytic activities, of P. gingivalis had strong positive correlations with clinical parameters of subjects who harbored the isolates. Therefore, the invasive ability of P. gingivalis is an important virulence factor, and the bacterial invasion step may be a good target for new therapeutics of periodontitis.

Keum Jin Baek

Department of Oral Microbiology and Immunology

School of Dentistry and Dental Research Institute, Seoul National University

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Keum Jin Baek

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Oral Microbiology and Immunology

School of Dentistry and Dental Research Institute, Seoul National University

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Department of Oral Microbiology and Immunology